The prostate gland is a hormone dependent tissue regulated throughout life by androgen action. It is also known that estrogens play key roles in prostate development and homeostasis and contribute to prostate carcinogenesis and progression. While estrogen actions through multiple receptors have been studied in detail in female organs, the mechanisms of estrogenic effects in the prostate gland are not well understood. Recently, our laboratory demonstrated that human prostate epithelial stem and progenitor cells from normal adult prostates express robust levels of estrogen receptors - ER, ER and GPR30 - and exhibit increased proliferative responses to estradiol-17. Further, preliminary data indicate heightened ER expression in human prostate cancer (PCa) stem-like cells suggesting that this minor tumor cell population may be a direct estrogen target. The overall goal of the proposed studies is to delineate the roles of estrogen receptors (ER, ER and GPR30) in epithelial stem and progenitor cells of the normal and cancerous human prostate gland and to elucidate their specific roles in promoting or preventing carcinogenesis and progression. Three Specific Aims are proposed to accomplish these goals. AIM 1: Define the specific roles for ER, ER and GPR30 in regulating self-renewal, amplification, apoptosis and/or differentiation in human prostate stem- progenitor cells from normal adult tissues. To accomplish this, we will utilize a fully characterized prostasphere system to interrogate stem and progenitor cells from primary prostate epithelial cultures of disease-free organ donors. AIM 2: Elucidate the expression and roles of specific ERs in human prostate cancer stem-like cells. Primary epithelial cultures of benign and PCa cells from patient specimens will be utilized to enrich for stem/progenitor cells by prostasphere assay. Results will be supported by interrogating ER actions in two immortalized human PCa stem-like cell lines, HPET and HuSLC. Co-culture studies with normal and cancer- associated fibroblasts will be used to define the estrogen-responsive stem cell niche in normal and cancerous human prostates. AIM 3: Delineate the actions of specific ERs in mediating hormonal carcinogenesis in vivo and regulating PCa growth and progression. An in vivo cell recombination graft model has been established using normal human prostate stem/progenitor cells or human PCa stem-like cells mixed with embryonic mesenchyme to generate chimeric prostate-like tissues with normal human prostate epithelium or prostate cancer. Targeted ER knockdown and selective estrogen receptor modulators (SERMs) will be tested to delineate estrogen actions and regulate PCa growth in vivo. Together, the present approaches will provide the first direct evidence for ER-specific actions in human prostate stem and progenitor cells. Importantly, use of fresh human prostate specimens from organ donors and PCa patients will grant translational relevance to the findings. The detailed molecular results supported by in vivo responses of prostate cancer growth to SERMs have potential to identify new therapeutic targets for management of PCa and inform future drug discovery strategies using novel small molecules that target stem cell ER actions.